1. Field of the Invention
The present invention relates to the isolation and characterization of a Neurospora crassa gene encoding for an essential activity in the co-suppression process and to uses and applications thereof in vegetal, animal and fungine fields.
2. Description of the Prior Art
The production of transgenic organisms is of large utility both in basic and applied biological research. The transgenic DNA is usually integrated in the genome and transferred as a Mendelian character. However, in various instances, the transgene introduction induces gene silencing phenomena (Flavell, R. B. 1994), i.e. the repression of the expression of the transgene itself and/or of one or more endogenous homologous genes.
The gene silencing can act at two levels: transcriptional (trans-inactivation) where transgenes contain sequences homologous to the silenced gene promoter (Vaucheret, 1993); and post-transcriptional (co-suppression) which requires homologies between coding regions (Flavell, 1994; Stam et al., 1997; Baulcombe, 1996).
Generally the silencing induced by a transgene requires an almost complete sequence homology (from 70% to 100%) between transgene and silenced gene sequences (Elkind, 1990).
In the Neurospora crassa filamentous fungus, during the vegetative phase, the presence of transgenes induces a post-transcriptional gene silencing phenomenon, named “quelling” (Cogoni et al., 1996).
By using the al-1 gene (albino 1) (Schmidhauser et al., 1990) as silencing visual marker, many features of the phenomenon have been discovered (Cogoni et al., 1996). Particularly the al-1 gene “quelling” in Neurospora is characterized in that: 1) the gene silencing is reversible further to the loss of transgene copies; 2) the reduction of mRNA basal level results from a post-transcriptional effect; 3) transgenes containing at least a region of 132 base pairs which is identical to the region encoding for the target gene are sufficient to induce the “quelling”; 4) the duplication of promoter sequences is ineffective to induce the silencing; 5) the “quelling” exhibits a dominant behavior in eterocarions containing both transgenic and untransformed nuclei, indicating the involvement of a molecule which is acts “in trans” among the nuclei; 6) the expression of an aberrant RNA transcribed by the transgenic locus is strictly correlated to silencing, suggesting that the “quelling” can be induced and/or mediated by a transgenic RNA molecule.
Therefore homologies between Neurospora silencing and plant co-suppression can be pointed out. The gene silencing in Neurospora is reversible, as result of transgenic copies instability during mitotic phase; in plants also the co-suppression reversion is associated with the reduction of transgene copy number, resulting from intra-chromosomal recombination during mitosis or meiosis (Mittelstein Scheid et al., 1994; Stam et al., 1997). Thus both in plants and in Neurospora the transgene presence is required to maintain the silencing. As in Neurospora, a decrease of the mRNA basal level of the silenced gene results from a post-transcriptional mechanism (Dehio and Schell 1994; van Blokand et al., 1994; de Carvalho et al., 1995). Furthermore to induce the “quelling”, transgenes must contain a portion of the silencing target gene coding sequence, being the promoter region ineffective. In plants coding regions with no promoter sequences can induce silencing (van Blokand et al., 1994) and, as in the “quelling”, promoters or functionally active gene products are not required for the co-suppression.
One of the similarities between “quelling” and co-suppression in plants is that both mechanisms are mediated by diffusion factors. In Neurospora eterokaryotic strains, nuclei wherein the albino-1 gene is silenced are able to induce the al-1 gene silencing of the other not transformed nuclei, all sharing the same cytoplasmic environment (Cogoni et al., 1996). In plants the presence of a diffusion factor results from the fact that the co-suppression is effective in inhibiting the replication of Tobacco Etch Virus (TEV), a RNA virus with an exclusively cytoplasmic cycle. The occurrence of highly diffusible factors, which are effective to mediate the co-suppression, has been demonstrated using the grafting technique in tobacco (Palaqui et al., 1997), showing that silenced tobacco plants are able to transfer the silencing to non-silenced plants through grafting.
The fact that “quelling” and co-suppression share all these features suggests that mechanisms involved in post-transcriptional gene silencing in plants and in fungi can be evolved by an ancestral common mechanism.
Recently gene inactivation phenomena resulting from transgene introduction have been disclosed in animals. In Drosophila melanogaster the location of a transgene close to heterochromatic centers results in a variegate expression (Wallrath and Elgin, 1995; Pirrotta, V., 1997). Similar expression profiles have been observed when the reference transgene is within tandem arrayed transposons, indicating that tandem repeats are effective to induce the chromatin condensation. (Dorer and Henikoff, 1994). Again in Drosophila Pal-Bhadra et al. (1997) have observed that the transgene introduction can lead to gene inactivation phenomena, similar to the co-suppression.
Gene silencing phenomena resulting from transegene sequence repeats have been disclosed recently in mammalians.
Garrick et al. (1998) produced mouse transgenic lines wherein 100 transgenic copies are present only in a locus and are directly tandem arrayed. The transgene expression has been disclosed to be inversely proportional to the number of occurring copies, indicating that silencing phenomena dependent on repeat copies are present also in mammalians.